1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family which has been modified to attenuate expression of a gene coding for small RNA (sRNA).
2. Brief Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or U.S. Pat. Nos. 4,346,170; 5,661,012 and 6,040,160).
Another way to enhance L-amino acid production yields is to attenuate expression of a gene or several genes which are involved in degradation of the target L-amino acid, genes which are responsible for diverting the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes which are involved in the redistribution of carbon, nitrogen, and phosphate fluxes, and genes coding for toxins etc.
Small, untranslated RNAs are present in many different organisms, ranging from bacteria to mammals. These RNAs carry out a variety of biological functions. Many of them can function as regulators of gene expression at the posttranscriptional level, either by acting as antisense RNAs, by binding to complementary sequences of target transcripts, or by interacting with proteins. Regulatory RNAs are involved in the control of a large variety of processes such as plasmid replication, transposition in pro- and eukaryotes, phage development, viral replication, bacterial virulence, global circuits in bacteria in response to environmental changes, or developmental control in lower eukaryotes (Argaman L. et. al., Current Biology, 11: 941-50 (2001)).
Small RNA (sRNA) molecules have gained much interest recently. Many Escherichia coli genes are known to code for sRNAs: c0067, c0293, c0299, c0343, c0362, c0465, c0614, c0664, c0719, csrB, dicF, dsrA, ffs, gadY, gcvB, is092, is102, is128, isrA, micC, micF, oxyS, rnpB, rprA, rybA, rybB, rydB, ryeA, ryeB, ryeC, ryeD, ryeE, ryfA, rygB, rygC, rygD, sgrS, spf, sraA, sraB, sraD, sraE, sraG, sraH, sraI sraJ, sraK, sraL, sroA, sroB, sroC, sroD, sroE, sroF, sroG, sroH, ssrA, ssrS, t44(tff), tp2, tpke11, tpke70 (Hershberg, R., et. al., Nucleic Acids Res., 31(7):1813-20 (2003) and Vogel, J., et al, Nucleic Acids Res., 31(22): 6435-43 (2003)). Most of these genes are still uncharacterized and their cellular roles are unknown. Traditionally, most RNA molecules were thought to function as mediators that carry the information from the gene to the translational machinery. Exceptions were the transfer RNAs and ribosomal RNAs that had long been known to have functions of their own, associated also with translation. However, it is now widely acknowledged that other types of untranslated RNA molecules (sRNA) exist that are involved in a diverse range of functions, from structural through regulatory to catalytic (Hershberg, R., et al., Nucleic Acids Res. 31(7): 1813-1820 (2003)).
The sraE and rygB genes encode small, untranslated RNAs-SraE and RygB of approximately 89 nt and 83 nt in length, respectively, which are encoded within the same inter-ORF region of the genome. Interactions between the SraE RNA and Hfq protein and between the RygB RNA and Hfq have been detected, SraE RNA and RygB RNA bound Hfq quite efficiently (>30% bound) (Wassarman, K. M. et al, Genes Dev. 1; 15(13):1637-51 (2001)). There is some sequence similarity between sraE and rygB, and they are transcribed in the same direction. SraE and rygB, which are located in the same intergenic region between aas and galR, show significant sequence similarity of 77% identity over 84 nt (Hershberg, R., et. al., Nucleic Acids Res., 31(7):1813-20 (2003)). Despite this high sequence similarity, these two sRNAs exhibit an almost mutually exclusive expression pattern: RygB levels increase around the onset of the stationary phase and decrease thereafter (Vogel, J., et al, Nucleic Acids Res., 31(22): 6435-43 (2003)), whereas SraE is produced as stationary phase progresses (Argaman, L. et al, Current Biology, 11: 941-50 (2001)).
The sroE gene encodes a small, untranslated RNA called SroE. SroE sRNA was shown to be processed from a longer transcript, that is, the upstream gcpE gene. Its 5′ end was mapped to the UAA stop codon of gcpE (third nucleotide). SroE extends into the promoter region downstream of the hisS gene. Both adjacent genes and the 110 bp IGR are conserved between E. coli and Salmonella species; the SroE sequences are predicted to fold into identical two-stem-loop structures with any sequence variation confined to the loops. The rybB gene is considered to be a genuine sRNA gene. RybB is produced as a shorter processed RNA species late in growth. The estimated half life was determined in stationary phase to be 8 min. The half life of SraE in stationary phase is 16 minutes; the half life of RygB in stationary phase is 30 minutes. SraH is one of the most stable known sRNA. The half life of SraH in stationary phase is 32 minutes (Vogel, J., et al, Nucleic Acids Res., 31(22): 6435-43 (2003)).
Expression of sraE is not affected by heat or cold shock treatment during early growth. The promoter of the sraE gene is found to be active in vitro, and the transcript length is similar to that observed in vivo. Expression of the E. coli K12 sraA gene was investigated in cells grown to different growth phases in either rich or minimal media supplemented with glycerol and in cells subjected to heat shock or cold shock treatment. The transcript levels of sraA were constant regardless of the conditions. SraB RNA is expressed during the stationary phase only and is at the highest levels at 8 and 10 hr after dilution of the culture. The gcvB gene is expressed in the early logarithmic phase, but its production slows with cellular growth. It was found that most of the gcvB transcripts read through the first terminator and stop at the second one, and thus result in an RNA product of 205 nucleotides. GcvB RNA is not affected by heat or cold shock treatment during early growth. Minor increases in GcvB expression were detected during the stationary phase when the cells were grown in glycerol minimal medium. SraH RNA is highly abundant during the stationary phase, but low levels can be detected in exponentially growing cells as well. Expression of sraH is not affected by heat or cold shock treatment during early growth. In vitro transcription of sraH resulted in a product of approximately 120 nucleotides, which corresponds to the predicted full-length RNA (Argaman, L. et al, Current Biology, 11: 941-50 (2001)). An interaction between RyhA (SraH) RNA and Hfq, a small, highly abundant RNA-binding protein, has been detected. High-copy expression of ryhA (sraH) causes increased expression of rpoS in minimal media (Wassarman, K. M. et al, Genes Dev. 1; 15(13):1637-51 (2001)).
The dsrA gene encodes DsrA RNA, a small (87-nt) regulatory RNA of E. coli that acts via RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (sigmas), and H-NS, a histone-like nucleoid protein and global transcription repressor (Lease R. A., et al, Proc. Natl. Acad. Sci. USA, 95(21):12456-61 (1998)). Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Genes induced by DsrA have been identified by using transcription profiling via DNA arrays (Lease R. A., et al, J. Bacteriol., 186(18):6179-85 (2004)). Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. Overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant, confirming that DsrA RNA plays a regulatory role in acid resistance.
Both the rate of transcription initiation of the dsrA gene and the stability of DsrA RNA are regulated by temperature, increasing at low temperature (Repoila F. and Gottesman S., J. Bacteriol., 183(13):4012-23 (2001)). The dsrA promoter is temperature-sensitive (Repoila F. and Gottesman S., J. Bacteriol., 185(22):6609-14 (2003)).
DsrA RNA acts by base-pairing to activate or repress translation, or to destabilize mRNAs. Base-pairing between this regulatory RNA and its target mRNAs requires the Sm-like Hfq protein, which most likely functions as an RNA chaperone to increase RNA unfolding or local target RNA concentration (Storz G., et al, Curr. Opin. Microbiol., 7(2):140-44 (2004)).
The rprA gene encodes a 106 nucleotide regulatory RNA called RprA. As with DsrA, RprA is predicted to form three stem-loop structures. At least two small RNAs, DsrA and RprA, participate in the positive regulation of the stationary phase sigma factor RpoS translation. Unlike DsrA, RprA does not have an extensive region of complementarity to the RpoS leader, leaving its mechanism of action unclear. It was assumed that RprA is non-essential in the positive regulation (Majdalani, N., et al., Mol. Microbiol, 39(5), 1382-94 (2001)).
The E. coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, both of which are the transcriptional regulators of the gcvTHP operon which encodes the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, which are periplasmic-binding protein components of two major peptide transport systems which are normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA-phoA and dppA-lacZ gene fusions. Although the mechanism(s) involved in the represssion by gcvB of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level. The sequence of gcvB was shown to contain two sites for transcription termination (M. L. Urbanowski et al, Mol. Microbiol., 37: 856-68 (2000)).
The micC gene (IS063) encodes a ˜100-nucleotide small-RNA transcript. The expression of this small RNA is increased at a low temperature and in minimal medium. Twenty-two nucleotides at the 5′ end of this transcript have the potential to form base pairs with the leader sequence of the mRNA encoding the outer membrane protein OmpC. MicC was shown to inhibit ribosome binding to the ompC mRNA leader in vitro and to require the Hfq RNA chaperone to function (Chen, S., et al., J. Bacteriol., 186(20):6679-80 (2004)).
The ryeE gene encodes a small, untranslated RNA-RyeE RNA 86 nt in length. All known sRNA are encoded within intergenic (Ig) regions (defined as regions between ORFs). The Ig region corresponding to ryeE is highly conserved when compared to the closely related Salmonella and Klebsiella pneumonia species. An interaction between RyeE RNA and Hfq protein has been detected, RyeE RNA bounds Hfq quite efficiently (>30% bound). Overproduction of RyeE causes decreased expression of rpoS during the stationary phase in LB (Wassarman, K. M., Genes Dev., 15(13): 1637-51 (2001)).
But currently, there have been no reports of inactivating a gene coding for sRNA for the purpose of producing L-amino acids.